The biological control of plant pathogens was detailed by
Van Driesche & Bellows (1996). It involves the ecological management of a
community of organisms. In the case of plant pathogens, however, there are
two distinctions from biological control of organisms such as insects and
plants. First, the ecological management occurs at the microbial level,
typically in microcosms of the ecosystem such as leaf and root surfaces
(Andrews 1992). Second, biological control agents include competitors, as
well as parasites. While hyperparasites of plant pathogens and natural
enemies of nematodes function in much the same way as do parasitoids, in
arthropod systems (by destroying the pest organisms), competitors function by
occupying and using resources in a nonpathogenic manner and in so doing
exclude pathogenic organisms from colonizing plant tissues. Microbes which
negatively affect pathogenic organisms are called antagonists.

Diseases of roots, stems, aerial
plant surfaces, flowers, and fruit are caused by a wide variety of pathogens.
Because of this diversity, the antagonist species, which negatively affect
plant pathogens and the mechanisms by which they accomplish their beneficial
action, are also quite varied. Their biological and taxonomic diversity is
covered in some detail in several texts and reviews, including Cook and Baker
(1983), Fokkema and van den Heuvel (1986), Campbell (1989), Adams (1990), and
Stirling (1991). This section briefly introduces the antagonists of some
important plant pathogens as representative of the broad taxa which are
important in this field, beginning with agents affecting microbial pathogens
of roots, and proceeding through pathogens of stems, leaves, flowers, and
fruit. Natural enemies of plant parasitic nematodes are treated in the last
section.

Root Pathogens

Root diseases are caused by a wide
variety of fungi, and by some bacteria, in many crops and plant systems.
Biological control agents recognized as significant in suppression of these
diseases are largely antagonists that can occupy niches similar to the
pathogens and either naturally or through manipulation out compete the
pathogens in these niches. Antibiotic production is also important in a few
cases, as are mycoparasitism and induced resistance. Streptomyces scabies,
the causative organism of potato scab, is suppressed by naturally
occurring populations of Bacillus subtilis, and saprotrophic Streptomyces
sp.). Other microorganisms recognized as suppressing fungal diseases
include species of Pseudomonas and Bacillus. Saprotrophic
Fusarium fungi are able to suppress populations of pathogenic Fusarium
spp. through competition for nutrients. There are few well-documented cases
of induced resistance for soil-borne pathogens, and these are mostly of wilt diseases.
Examples of organisms that induce resistance in plants to pathogens include
nonpathogenic strains of Fusarium spp. Verticillium spp. &
Gaeumannomyces spp. Mycoparasitic flora such as Anthrobotrys pp,
Coniothyrium minitans Campbell and Sporidesmium scerotivorum
Uecker et al., can be added to soil against fungal diseases. Bacillus spp.
and especially Pseudomonas spp. are among bacteria that have
properties particularly suited to effective suppression of root-infecting
pathogens in soil,such
as antibiotic production and competition for Fe3+ ions. Mycetophagous soil
amoebae have also been noted feeding on pathogenic fungi. These amoebae
generally require moist conditions in which to function, and may be important
in the natural control of some fungi.

Stem Pathogens

Diseases of plant stems produce
symptoms which include decay and cankers on forest and orchard trees. and
such wilts @ts I)utch elni disease and chestnut blight (caused by the fungus Cryphonectria
parasitica (Murrill) Barr of Asian origin infecting the American
chestnut. Castanea dentata [Marshaml Borkjauser). Because the
etiologies of stem diseases vary, the taxa involved in biological control
also vary. In many stem diseases, the pathogen colonizes a part of the host
which initially is relatively free of microorganisms, such as a pruning
wound. Successful biological control in such circumstances depends on rapidly
colonizing this pristine environment with a nonpathogenic antagonistic
competitor (VanDriesch & Bellows 1996). Primary among these are
competitively antagonistic fungi, including saprotrophic members of the
genera Fusarium, Cladosporium, Trichoderma, and Phanerochaete, and
such antibiotic-producing bacteria as Bacillus subtilis and Agrobacterium spp.
In the case of chestnut blight, hypovirulent strains of the pathogen itself
are crucial in bringing about biological control. In this case, hypoviruience
is transmitted cytoplasmically to virulent strains already infecting trees,
and disease symptoms decline and disappear (VanDriesch & Bellows 1996).

Leaf Pathogens

The growth of microorganisms on
leaves is normally severely restricted by environmental factors. Nutrient
levels generally are low on leaf surfaces, and microclimate variables,
especially leaf surface moisture, temperature, and irradiation, are often
unfavorable for microbial development. In temperate climates and arid
tropical regions, water will be intermittent on leaf surfaces, but may be
continually present in humid tropical regions. Temperatures on leaf surfaces
exposed to direct radiation may rise to several degrees above ambient, The
result of such variation is that microbial floral development on leaf
surfaces varies from general scarcity in temperate climates to more extensive
microbial films in tropical rain forests (Campbell 1989).

Microbes that most frequently are
recorded as saprotrophs on surfaces of crop plants in temperate conditions
and, therefore, the species which are candidates as antagonists of pathogens,
include the fungi Aureobasid m pullulans (de Bary) Arnaud, Cladosporium
spp., and such yeasts as Cr.yptococcus spp. and Sporobolomyces spp.
Beneficial bacteria in the phyllosphere include members of such genera as Erwinia,
Pseudomonas, Xanthomonas, Chromobacterium and Klebsiella. These
lists,based on microbial surveys, usually give no indication of activity of
the organisms, but this information can be obtained from experimental
studies. For example, early studies on control of botrytis rot in lettuce
(Wood 1951) indicated that several organisms were successful in suppressing
the disease when sprayed on lettuce (Lactuca sativa L.) plants, among
them Pseudomonas sp., Streptomyces sp., Trichoderma viride Persoon:
Fries, and Fusarium sp. Similar studies show varying degrees of
effectiveness in other cropping systems (Peng & Sutton 1991; Sutton &
Peng 1993a,b; Zhang et al. 1994). The microbial composition and biological
activity of phylloplane microbes can vary with season, position on the top or
bottom of the leaf and on location in the plant canopy, depending on the
degree of exposure relative to prevailing winds and rain (Campbell 1989).

Nonpathogenic species of the
fungal genus Colletot richum 1981; Dean & Kuc 1986) can be used to
induce resistance in cucumbers against pathogenic species of the same genus.
Inoculation with a nonpathogenic strain of a virus confers protection to
plants from pathogenic strains in many diseases. The bacterium Bdellovibrio
bacteriovorus Stolp & Starr is aparasite of pathogenic bacteria.
Finally, there are numerous parasitic fungi that attack pathogenic fungi
(Kranz 1981). Among those that have been studied in detail, principally as
agents against leaf rusts and mildews, are Spbaerellopsis filum (Bivona-Bernardi
ex Fries) Sutton, Verticillium lecanii (Zimmerman) Viegas, and Ampelomyces
quisqualis Cesati ex Schlechtendal (VanDriesch & Bellows 1996).

Flower & Fruit Pathogens

Flowers are ephemeral structures
and as such have limited opportunity to become infected. One major disease of
flowers which has received attention is fire blight of rosaceous plants,
caused by the bacterium Erwinia amylovora (Burril) Winslow et al.
Biological suppression of the disease has been achieved through use of the
nonpathogenic species Erwinia herbicola (Lohnis) Dye (Beer et al.
1984; Lindow 1985b), sometimes in combination with Pseudomonas syringae van
Hall. rwinia herbicola was
used successful by spraying aqueous suspensions of it onto the flowers just
before the time of potential infection (Campbell 1989). The mode of action is
primarily competitive exclusion, with the antagonist competing with the
pathogen for a growth limiting resource and possibly other effects such as
induced cessation of nectar secretion or accumulation of a host toxin (Wilson
and Lindow 1993a).

The diseases attacked through
biological control include diseases of fruit on the plant and post-harvest
diseases. One of the first systems developed was against Botrytis cinerea
Persoon Fries in vineyards, where sprays with spore suspensions of the
antagonist Ttrichoderma barzianum Rifai were effective in suppressing
disease incidence. Several organisms, including Gliocladium roseum,
Penicillium sp., Trichoderma viride, and Colletotrichum
gloeosporioides were as effective as fungicides in suppressing B.
cinerea on strawberries (Peng and Sutton 1991). A number of other
examples also have been reported (Sutton and Peng 1993a).

Post-harvest diseases, which can
be responsible for 10-50% loss of produce (Wilson and Wisniewski 1989;
Jeffries and Jeger 1990), have received considerable attention. Numerous
reports deal with suppression of post-harvest disease in fruit crops
(Campbell 1989; Wilson and Wisniewski 1989; Jeffries and Jeger 1990) by such
organisms as species of Penicillium, Bacillus, Trichoderma, Debaryomyces,
and Pseudomonas. The mode of action of many of these is generally
antagonism, often through the production of antibiotics, which reduce the
longevity, and germination of spores of pathogens. Others appear to suppress
pathogen growth through nutritional competition or induction of host
resistance (Wilson and Wisniewski 1989). Postharvest rots include major
diseases caused by Botrytis cinerea, Rhizopus spp., and other fungi in
several crops. Competitive and parasitic fungi, including Tiichoderma
spp., Cladosporium herbarum (Persoon: Fries) Link and Penicillium
spp., give control as good as commercial fungicides. Enterobacter cloacae (Jordan)
Hormaeche and Eduards reduces rots liy Rhizopus spp., but there are
restrictions in its use on uncooked food products (Van Driesche & Bellows
1996).

Plant-parasitic Nematodes

Plant-parasitic nematodes inhabit
many soils and attack the roots of plants. They are affected by a range of
natural enemies, including bacteria, nematophagous fungi, and predacious
nematodes and arthropods. There is some limited evidence for virus
association with nematodes (Loewenberg et al. 1959), but the etiology of
these viruses is not well known (Stirling 1991). The biologies of natural
enemies of nematodes were reviewed by Sayre and Walter (1991) and Stirling
(1991).

Bacteria That Affect Plant-Parasitic
Nematodes

A few bacterial diseases of
nematodes have been reported (Saxena and Mukerji 1988); other bacteria
produce compounds that are detrimental to plant-parasitic nematodes (Stirling
1991). The most widely studied of the bacterial pathogens of nematodes are in
the genus Pasteuria. Early work was focused on Pasteuria penetrans (Thorne)Starr and Sayre. Recent evidence indicates that this taxon represents an
assemblage of numerous pathotypes and morphotypes, and probably represents
several taxa (Starr and Sayre 1988). This bacterium has been found infecting
a large number of nematode species (more than 200 in about 100 genera, Sayre
and Starr 1988; Stirling 1991), does not attack other soil organisms, and is
the most specific obligate parasite of nematodes known. Its spores attach to
and penetrate the nematode cuticle. Most attention has been centered on
populations (Pasteuria penetrans sensu stncto, Start and Sayre 1988)
that attack root-knot nematodes (Meloidogyne spp.). The spores of P.
penetrans germinate a few days after a contaminated nematode begins
feeding on a root (Sayre and Wergin 1977). The bacterium reproduces
throughout the entire female body, and the female may either be killed or may
mature but produce no eggs. Bacterial spores (about 2 million from each
infected nematode, Mankau 1975) are released when the nematode body decomposes,
and they remain free in the soil until contacted by another nematode. They
tolerate dry conditions and a wide range of temperatures, and may remain
viable in the soil for more than six months. Because it is an obligate
parasite, it has not yet been possible to develop in vitro culturing
techniques for this bacterium. Different populations of the bacterium show
varying degrees of specificity to small numbers of nematode species, but the
mechanisms and degree of specificity remain to be elucidated (Stirling 1991).
Pasteuria penetrans appears responsible for some cases of natural
regulation of nematode populations (Sayre and Walter 1991).

Some strains of Bacillus
tburingiensis are also known to have activity against nematodes,
including plant-parasitic species. Zuckerman et al. (1993) report efficacy of
a strain against Meloidogyne incognita (Kofoid and White) Chitwood, Ratylencbus
reniformis Linford and Oliveira, and Pratylenchus penetrans Cobb
in field and glasshouse trials. The body openings of these nematodes are too
small to permit the ingestion or other ingress of the bacterium, and
Zuckerman et al. (1993) suggest that the mode of action is either a beta
exotoxin (Prasad et al. 1972; Ignoffo and Dropkin 1977) or a delta endotoxin
released following bacterial cell lysis. A strain of B. thuringiensis with
a nematotoxic delta endotoxin is the subject of a European Patent Application
by Mycogen Corporation of San Diego, California (Zucherman et al. 1993).

Fungi That Affect Plant-Parasitic
Nematodes

Many fungi attack nematodes in the
soil (Barron 1977; Stirling 1991). Numerous species have been reported from
all types of soils. The taxonomy of the group has been subject to revision,
and the generic names recognized in Stirling (1991) are used here (Van
Driesche & Bellows 1996).

Some nematophagous fungi are
endoparasitic in nematodes. Among these are genera which reproduce through
motile zoospores (e.g., Catenaria anguillulae Sorokin, Lagenidium caudatum
Barron, Aphanom.yces sp.), which generally appear only weakly
pathogenic in healthy nematodes (Stirling 1991). Other endoparisitic fungi
possess adhesive conidia, and the infection process begins when conidia
adhere to a nematode's cuticle (e.g., the genera Vellicillium, Drechmeria,Hirsutella,
Nematoctonuss). In Nematoctonus spp.,the germinating spores
secrete a nematotoxic compound which causes rapid immobilization and death of
nematodes (Giuma et al. 1973). A few species (Catenaria auxila [Kuhn]
Tribe, Nematophthora gynophila Kerry and Crump) parasitize adult
females or nematode eggs rather than juveniles.

Other fungi capture nematodes
through use of special trapping stmctures, and have been termed
"predatory." Among the more common of these fungi are species in
such genera as ,Monacrosporium, Arthrobotrys, and Nematoctonus. These
fungi consist of a sparse mycelium, modified to form organs capable of
capturing nematodes. These organs include adhesive structures, such as
adhesive hyphae, branches, knobs, or nets (Stirling 1991). There are also
nonadhesive rings, the cells of which expand when touched on their inner
surface, constricting the interior of the ring and trapping nematodes. Most
of these fungi are not specific and attack a wide range of nematode species.
They are widely distributed (Gray 1987, 1988) and most are capable of
saprotrophic growth, but often appear limited in this phase in the soil. Many
soils suppress the growth of these fungi (a condition called soil fungistasis
or mycostasis). This is possibly due to two different causes. Mankau (1962)
concluded that a water-diffusible substance was responsible for inhibited
germination in tests of soil from southern California (U.S.A.). Other studies
have indicated increased activity following soil amendments with nutrients
(Olthof and Esrey 1966) or organic material (Cooke 1968), which implies
fungistasis may be a result of resource limitation. Following saprotrophic
growth, formation of trapping structures occurs which is apparently stimulated
by nematodes (Nordbring-Hertz 1973; Janssen & Nordbring Hertz 1980).
Stirling (1991) suggests that this phase of predacious activity is followed
by diversion of resources to reproduction, followed by a relatively dormant
phase (Van Driesche & Bellows 1996).

Other fungi are facultatively
parasitic on nematodes. Of the few of these fungi that are significant
pathogens of root knot and cyst nematodes, Verticillium spp, are among
the most important. These fungi can parasitize nematode eggs, and Verticillium
chlamydosporium Goddard plays a major role in limiting multiplication of Heterodera
avenae Wollenweber in English cereal fields (Kerry et at. 1982a,b). Paecilomyces
lilacinus (Thom) Samson parasitizes eggs of Meloidogyne incognita (Jatala
et al. 1979) and Heterodera zeae Koshy, Swarup, and Sethi (Dunn 1983;
Godoy et al. 1983). Dactylella oviparasitica Stirling and Mankau, a
parasite of Meloidogyne eggs, is thought to be at least partially
responsible for the natural decline of root-knot nematodes in Californian
peach orchards (Stirling et al. 1979).

Predacious Nematodes That Affect
Plant-Parasitic Nematodes

Predatory nematodes are found in
four main taxonomic groups: Monochilidae, Dorylaimidae, Aphelenchidae and Diplogasteridae.
Each possesses a distinct feeding mechanism and food preferences (Stirling
1991). The monochilids have a large buccal cavity that bears a large dorsal
tooth; all species are precdacious, feeding on protozoa, nematodes, rotifers,
and other prey, which may be swallowed whole, or pierced and the body
contents removed. The dorylaimidss are typically larger than their prey and
possess a hollow spear which is used either to pierce the body of the prey or
to inject enzymes into the food source and suck out the predigested contents.
The group is considered omnivorous. but the feeding habits are known only for
a few species (Ferris and Ferris 1989). Almost all the predatory aphelenchids
are in the genus Seinura. Although small, they can feed on nematodes
larger than themselves by injecting the prey with a rapidly paralyzing toxin
through their stylet. The diplogasterids, typically a bacteria-feeding group,
have a stoma armed with teeth, and the species with large teeth prey on other
nematodes. Species in all these groups are generally omnivorous, feeding on
free-living as well as plant parasitic nematodes. The role of individual
species in the population dynamics of plant parasitic nematodes in the soil has
been difficult to quantify, but it is possible that a number of species
may act together to produce a significant impact (Stirling 1991).

Insects and Mites

Several microanthropods in the
soil, including mites and Collembola, prey on nematodes, and high predation
rates have been recorded in vitro (Stirling 1991). A few genera are
obligate predators of nematodes, while other genera are more general feeders
and consume nematodes as well as other foods (Moore et al. 1988; Walter et al. 1988; Sayre and
Walter 1991). The information available suggests that as a group,
microanhropods are probably significant predators on nematodes in some soils
and habitats. However, limited information about predation rates in soil is
available, and more work is required to assess the impact of this group on nematode
populations.

VanDriesch &
Bellows (1996) concluded that this overview touched briefly on groups of
organisms which are antagonistic to plant pathogens and nematodes. These
antagonists vary both in their innate ability to suppress plant pathogens and
in their ability to thrive and compete in different environments.
Consequently the selection of an organism or organisms for any particular
biological control program is a compromise among these parameters and
abilities. In addition, the selection of organisms depends on the approach
taken for their use (inoculative augmentation, inundative augmentation, or
natural control through conservation.

Organisms for biological control
of plant disease can be used in various ways, but most attention has been
given to their conservation and augmentation in a particular environment,
rather than to the importation and addition of new species as is often done
for insect or weed control. The choice of these approaches is in part because
there is usually a diverse set of microbes already associated with plants.
These microbes provide substantial opportunity for development of resident
species as competitors or antagonists to pathogenic organisms. Both
conservation and augmentation have some application in each of the main
groups of plant diseases. The use of microbes for control of plant pathogens
is covered in more detail in several texts, including Cook and Baker (1983),
Parker et al. (1983), Fokkema and van den Heuvel (1986), Lynch (1987),
Campbell (1989), and Stirling (1991) and in other review articles (Wilson and
Wisniewski 1989; Adams 1990; Jeffries and jeger 1990; Sayre and Walter 1991;
Andrews 1 92; Cook 1993; Sutton and Peng 1993a).

Plant pathogens are attacked with
biological control through conservation is accomplished either by preserving
existing microbes which attack or compete with pathogens or by enhancing
conditions for their survival and reproduction at the expense of pathogenic
organisms. Conservation is applicable in situations where microorganisms
important in limiting disease causing organisms already occur, primarily in
the soil and plant residues but in some cases also on leaf surfaces. They may
be conserved by avoiding practices which negatively affect them (such as soil
treatments with fungicides). The soil environment may be enhanced for some
beneficial organisms through adding organic matter, such as soil amendments
(Van Driesche & Bellows 1996).

Biological control of plant
pathogens through augmentation is based on mass culturing antagonistic
species and adding them to the cropping system. In the context of the
examples discussed in this text, this is augmentation of natural enemy
populations, because the organisms used are usually present in the system,
but at lower numbers or in locations different than desired. The purpose of
augmentation is to increase the numbers or modify the distribution of the
antagonists in the system. In some cases, such organisms are taken from one
habitat (for example the soil) and augmented in another (for example the
phyllosphere). Tire activity of augmenting microbial agents is sometimes
termed "introduction" in the plant pathology literature, in the
sense of "adding@
them to the system (Andrews 1992; Cook 1993). However, he organisms
introduced are usually found in a local ecosystem and are not introduced from
another region of the world.

Augmentation of antagonists
naturally involves two approaches. The first is direct augmentation, at
potential infection sites or zones, with organisms antagonistic or parasitic
to the pathogens themselves. In this approach, the antagonist population is
directly responsible for disease suppression. A second approach is to
inoculate plants with nonpathogenic organisms that prompt general plant
defenses against infection by pathogens (induced resistance). Disease control
is then achieved through greater plant resistance to infection.

Substantial work has been done to
characterize the role of microorganisms in biological control of plant
diseases. The biological mechanisms underlying the success of these
antagonists in such settings may include initial competition for occupancy of
inoculation sites, competition for limiting nutrients or minerals, antibiotic
production, and parasitism (Van Driesche & Bellows 1996).

Understanding the principles that
apply to biological control of plant pathogens, the ecology of the system is
considered at the level of the pathogens and the agents used for control.
Aerial plant surfaces, usually present hostile environments to colonizing
microbes, in many cases consisting of surfaces protected by cuticular waxes,
with very small amounts of nutrients available on these surfaces. Further,
surfaces of the aboveground portions of plants may be dry. Consequently,
pathogenic microbes attempting to colonize these surfaces may face a number
of difficulties, including competition with other, nonpathogenic, microbes.

The rliizosphere (the roots and
the region immediately adjacent) is a somewhat richer environment than the
phyllosphere because of simple sugars, amino acids, and other materials
exuded by the roots, but in the remainder of the soil the growth of microbes
is often carbon limited (Campbell 1989), Moisture in the rhizosphere may be
more continuous in time and space than on the above ground surfaces of plants
(the phylloplane), but the rhizosphere may be subject to periodic drying.

Some forms of competition in these
environments are important to the ability of any particular organism to
increase in numbers and consequently to reduce the numbers or activity of
other organisms, including plant pathogens (Campbell 1989; Andrews 1992).
Microbial competition can be important at two main stages of growth of
pathogen populations. First, there may he competition during initial
establishment on a fresh resource that was not previously colonized by
microorganisms. Second, after initial establishment, there is further
competition to secure enough of the limited resources present to permit
survival and eventual reproduction. Microorganisms show many traits that may
characterize them as particularly adept at either the colonization phase or
subsequent phases of competition. Species referred to as r strategists
(ruderal species) have a high reproductive capacity. These species produce so
many spores or reproductive bodies that there is a high likelihood that some
will be found near any newly available resource. These species are
effectively dispersed and establish readily in disturbed habitats or in the
presence of noncolonized resources. They are found in disturbed settings
where easily decomposable organic matter or root exudates are found, and
where initial resource capture is crucial for survival. In contrast to these
r-strategists, species found in more stable situations face competition for
space and limited resources (Begon et al. 1986). These organisms, termed K-strategists,
become more dominant as a community matures and becomes more crowded. These
concepts form the endpoints of a continuum, and there are varying degrees of
r- and K-related characteristics in different microbes in various habitats
(see Andrews and Harris [19851 for further discussion on these concepts in
microbial ecology).

Plant pathogens are spread across
this r-K range of characteristics and vary in other important biological
characteristics (Van Driesche & Bellows 1996). There are opportunistic
pathogens that are able to attack young, weakened, or predisposed plants, but
may be poor competitors (Botrytis, Pythium, Rbizoctonia). There are
pathogens that tolerate environmental stresses. These organisms often live in
situations with few competitors, because few species are able to exist in
such environments. Some pathogens, such as the Penicillium species
that cause postharvest rots, produce antibiotics that inhibit competitors.
Other species (such as Fusarium culmorum [Smith] Saccardo) have a very
high competitive ability. It is important to understand the ecology of a
target pathogen before one can effectively consider what biological control
strategy might be most effective. Stress-tolerant and competitive species,
for example,require different biological control strategies and agents than
ruderal ones.

Similar to the way that
antagonists of plant pathogens vary in r-K and other characteristics, the
properties of an effective biological control agent will depend on the
setting in which it is intended to function, in many agricultural settings,
disturbance makes new resources available to microbes through crop residue
burial, cultivation, or planting. A frequent need, therefore, is a control
agent that has the characteristics of an r-strategist (Campbell 1989), which
can grow quickly and colonize new resources rapidly, with minimal nutrient
and environmental restrictions. It should function well in disturbed
environments and have some means (such as spores) of surviving in the soil or
on the plant near to the pathogen inoculum or the Source or site of
infection. Biological control agents that are r-strategists are an
approximate equivalent of a protectant fungicide, being in place before the
pathogen infection cycle can begin. In other programs, such as those directed
against a pathogen which has already invaded the plant host, a more
competitive species will be required. Finally, a biological control agent may
have to be tolerant of abiotic stresses, particularly for use in dry climates
or on leaves.

Although there is much variation in soil types in different
locations, soils are typically rich in microflora, with propagules numbering
in the hundreds of thousands per gram of soil (Campbell 1989). In most soils,
growth of microorganisms is carbon-limited. either because what carbon is
available is not physically accessible or because the microbes do not possess
the enzymes necessary to degrade the carbon-containing molecules that are
present. An exception to this general limitation is the region immediately
surrounding plant roots. This region, the rhizosphere, contains easily
metabolized carbon and nitrogen sources such as amino acids, simple sugars,
and other compounds exuded by the roots. Consequently, this region is more
favorable than surrounding soil for the support of microflora. Root pathogens
and plantparasitic nematodes may be found growing on or in roots, but many
microbes in the soil will be dormant because of resource limitations. Because
there are many dormant organisms in the soil prepared to take advantage of
any favorable period or opportunity, competition for resources in the soil
may be significant and may limit the ability to augment beneficial organisms
and have them flourish, unless soils are first sterilized to eliminate
potential competitors. Therefore, much research surrounding biological
control of root diseases and nematodes has centered around identifying soils
which are naturally suppressive to particular disease organisms and
investigating the microbial components of the soil responsible for the
suppression. Management of such antagonistic organisms for biological control
can range from treatment of soil to favor the desirable organisms
(conservation) through inoculation of soils or plants with specific
beneficial microorganisms (augmentation) (Van Driesche & Bellows 1996).

The phyllosphere is significantly
different from the rhizosphere in its structure, ecology, nutrient
availability, and exposure to climatic factors (Andrews 1992). Leaves are
relatively hostile to microorganisms. They are generally hydrophobic and
covered with chitin and wax, which limits the amount of exudate (and hence
nutrients) that reaches the leaf surface. These and other factors impose
severe environmental restrictions to microbial growth on leaf surfaces.
Fungal pathogens of leaves often enter the leaf tissue very shortly after
germination of the pathogen and, consequently, are protected inside the plant
for much of their growth. Bacterial pathogens may multiply on the leaf surface
before invading leaf tissues. Biological control of disease can take place
either through general inhibition and competition on the leaf surface prior
to invasion of leaf tissues or through suppression of the disease after the
pathogen has invaded. Biological control within leaf tissues can occur
through one of several mechanisms, including induced resistance in the plant
and hyperparasitism of the pathogen. Woody stems are habitats low in
nutrients and often difficult for pathogens to penetrate, Because the wood
itself supports very few saprotrophic microorganisms, pathogens colonizing
the wood through wounds, dead branches, or roots find very few competitors.
Because there are few organisms present to conserve, protection of the wood
from these decay organisms can be achieved by protecting the relatively
small, well-defined wound or branch stub through inoculation (augmentation)
with specific microorganisms. These wounds are initially very low in sugars
or other nonstmctural carbohydrates, and antagonists such as Trichoderma spp.
can successfully compete for these limited resources. Many of the organisms
used in the biological control of stem diseases are employed by applying them
directly to stem wounds, where they colonize resources and subsequently exclude
pathogenic forms. This initial occupancy by antagonists subsequently limits
infection by decay-causing organisms, and hence controls the succession of
microorganisms in the wood. Of the successful, commercially-available
biological control products for plant diseases, several are for diseases of
woody stems (Campbell 1989).

There are several different ways
in which a microbial biological control agent can operate against a targeted
plant pathogen (Elad 1986). Among these are competition, induction of plant
defenses, and parasitism.

Some agents act through
competition for limited resources, and through this competition the growth of
the pathogen population is suppressed, reducing the incidence or severity of
disease. One important component of competition can be competition for Fe-31
ions. Chemicals called siderophores, which are produced by many species of
plants and microbes, sequester these ions. Highly efficient siderophores from
nonpathogenic microbes can remove Fe-3l ions from the soil, outcompeting
siderophores from pathogens and thereby limiting the growth of pathogen
populations. Some biological control agents compete through the production of
antimicrobial substances such as antibiotics which inhibit the growth of
pathogens directly, rather than by preemptive consumption of limiting
resources.

An important mechanism limiting
infection is the induction of plant defenses against pathogensby
nonpathogenic organisms. Cross-protection and induced resistance are
mechanisms in which plants are intentionally exposed to certain
(nonpathogenic or mildly pathogenic) microbes, thereby conferring in the
treated plants some resistance to infection by pathogens. induced plant
defenses may include lignification of cell walls through the addition of
chemical cross-linkages in cell wall peptides which makes the establishment
of infection through lysis more difficult, suberification of tissues (where
plant cell walls are infiltrated with the fatty substance suberin, making
them more corklike), and other general defenses, including production of
chitinases and Beta 1,3-glucanases. These plant defenses then limit later
infection by pathogens. The biological control agent employed may be an
avirulant strain of the pathogen, a different forma specialis, or even
a different species of microorganism.

A third mechanism by which
beneficial microorganisms suppress plant pathogens is parasitism. Some
species of Tricboderma, for example, attack pathogenic fungi, leading
to the lysis of the pathogen. Natural enemies of plant-parasitic nematodes
include bacterial diseases and nematophagous and nematopathogenic fungi.

As is the case of conservation of
natural enemies of pest arthropods and weedy plants, conservation activities
for the suppression of plant pathogens consist of either avoiding practices
which reduce desirable antagonists or actively modifying the environment to
favor or selectively enhance the growth of such species. in the case of soil
microflora, species employed for biological control of plant pathogens are
often competitive antagonists. Adding amendments to soil is one way in which
soil microorganisms may be managed to enhance populations of these beneficial
organisms. Addition of organic matter to soils for control of Streptomyces
scabies, the causative organism of potato scab, is one example. Addition
of carbon sources to soil increases general microbial activity that leads to
reductions in S. scabies. Specifically, Bacillus subtilis and
saprotrophic species of Streptomyces were encouraged by barley,
alfalfa, or soy meal (Campbell 1989). Soy meal was also a substrate for
antibiotic production against S. scabies. A general rise in soil
organic matter also gave control of Phytophthora cinnamomi Rands in
avocado in Australia (Manajczuk 1979). The addition of more than 10 tons of
organic matter per hectare per year led to general increases in numbers of
bacteria. Lysis of the hyphae and sporangia of the pathogen were attributed
to species of Pseudomonas, Bacillus, and Streptomyces.

Some soils appear to suppress
disease naturally and may contain antagonistic or antibiotic flora which
flourish without the need for amendments. An example of such suppressive
soils is the Fusarium-suppressive soil in the Chateaurenard District of the
Rhone Valley in France Here, Fusarium oxysporum f: sp. melonis Snyder
and Hansen is present, but no disease develops when susceptible melon varieties
are grown. These soils are suppressive for several other types of F.
oxysporum, but not to other species or genera of pathogens. The
suppressive nature of the soils is clearly biotic, because the soils lose
their suppressive ability when steam-sterilized, and the suppressive ability
can be transfered to other soils. The antagonists principally responsible for
this suppression are nonpathogenic strains of F oxysporum and F.
solani (Martius) Saccardo. The suppression appears to be due to
fungistasis induced by nutrient limitation. The competing fungi appear to
have nearly the same ecological niche as the pathogenic forms, and the
saprotrophic forms outcompete the pathogens for limiting resources so that
dormant chlamydospores of the pathogen do not germinate in the presence of
host root exudates. It may be possible to develop systems for other areas
using the antagonists from the Chateaurenard area (Campbell 1989), although
additional research may be necessary to permit their effective operation in different
soils. Other soils suppressive to Fusarium wilts are known. There are
numerous other examples of suppressive soils, although some soils or
combinations appear to give somewhat variable results (Van Driesche &
Bellows 1996).

The conservation of existing flora may be important in
limiting the extent of a number of leaf diseases (Campbell 1989). These
effects are often revealed through the use of fungicides, which deplete
extant fungi, permitting the development of previously unimportant diseases,
Fokkerna and de Nooij (1981), for example. evaluated the effects of various
fungicides on leaf surface saprotrophs that have been used in biological
control. Wide-spectrum fungicides allowed almost no growth of saprotrophs,
while more selective agents permitted some growth of several genera of
saprotrophs. in cases where these saprotroph populations play an important
role in limiting disease organisms, the application of fungicides would
eliminate their contribution to pathogen suppression. A case illustrated by
Fokkerrrt and de Nooij (1981), is where plants treated with benomyl (a
systemic fungicide) had fewer saprotrophs and developed more necrotic leaf
area when inoculated with Cocbliobolus sativus (Ito and Kuribayashi)
Drechsler ex Dastur than nontreatect plants (C. sativus is insensitive
to benomyl), Another example (Mulinge & Griffiths 1974) is leaf rust of
coffee (Coffea arabica L.), caused by Hemileia vastatrix Berkeley
and Broome. The disease can be controlled by proper application of fungicide.
However, if fungicides are applied in one year and not in the next, the
disease is worse on the treated plants than on those which did not receive
treatments either year. The elimination of the saprotrophic flora by the
fungicide removes their natural suppressing influence on the disease
organisms, permitting the disease to worsen , Here, careful use of selective
fungicides are crucial to conserving the important antagonistic flora and
permitting their beneficial action (Van Driesche & Bellows 1996).

Several reports exist of
substantial natural control (control by natural enemies without intentional
manipulation) of plant-parasitic nematodes. Stirling (1991) and Sayre and
Walter (1991) review several of these; one example is that of the natural
suppression of the cereal cyst nematode Heterodera avenae in cereal
cultivation in the Great Britain (Gair et al 1969). In this case, populations
of the nematode initially increased for the first 2-3 years of cultivations,
and then declined continually during 13 years of continuous cultivation of
both oats and barley (a more susceptible crop). Four species of nematophagous
fungi were present in the soil. The two species principally responsible for
nematode suppression were Nematopbtbora gynophila and Verticillium
chlamydosporium. Both fungi attacked female nematodes, either destroying
them or reducing their fecundity. The activity of both fungi was greatest in
wet soils during laboratory trials (Kerry et al. 1980). Although natural
suppression of the nematode population required some time to develop in these
soils, but once established it maintained the population below the economic
threshold (Stirling 1991).

Conserving nematode antagonists in
soils (as opposed to directly enhancing their numbers), is a matter that has
received relatively little attention (Van Driesche & Bellows 1996). The
application of toxins (insecticides, fungicides) to aerial portions of crops
or directly to soils often leads to pesticide activity in the soil. All
nematicides are nonselective in their action and, hence, will kill predatory
nematodes (Stirling 1991). In addition, herbicides have well-documented
effects on soil microorganisms (Anderson 1978) and may well exert some influence
on microbial antagonists of nematodes, and insecticides may negatively affect
soil microarthropods. Many fungicides are known to be detrimental to
nematophagous fungi (Mankau 1968; Canto-Saenz and Kaltenbach 1984; Jaffee and
McInnis 1990), but at levels higher than would be expected under normal field
practice. Among the fumigant nematicides, ethylene dibromide (EDB) and
eibromo-chloro-propene (DBCP) appear nontoxic to the nematode-trapping fungi
(Mankau 1968), and several herbicides were shown to be unharmful to Arthrobotrys
sp. (Cayrol 1983). Despite these potentially significant effects on
beneficial microflora and fauna and the possibility of conserving these
organisms by appropriate choice of material, little has emerged to integrate
these ideas into normal farming practice (Van Driesche & Bellows 1996).
Perhaps because there has been no serious emergence of nematode problems
associated with the use of these materials, this statusquo is
justified. Nonetheless, the opportunities for conserving biologically
important agents should be considered in the development of future integrated
management programs for plant-parasitic nematodes. Similarly, cultivation
practices may be selected to favor natural enemies of nematodes, Among these
are minimum or conservation tillage, which reduced the number of cysts of Heteroderci
avenae on roots and the amount of damage caused by the nematode on wheat
in Australia (Roger and Rovira 1987). other practices which may affect
populations of natural enemies include normal tillage (which adds crop
residue to the soil and thus may favor certain beneficial organisms) and crop
rotation sequences (Stirling 1991). The knowledge that some soils are
naturally suppressive to nematodes prompts the question of whether or not the
features of these soils can be used to improve biological control. in all
documented instances where they have been studied, the suppressive properties
of these soils appear to result primarily from the action of one or two
specific biological control agents (Stirling 1991). The suppressiveness
requires substantial time to develop, and considerable crop loss might be
incurred during such an initial phase. Some risk is involved also, because
the suppressive nature of the soil may not develop to suitable levels.
Careful management of crop varieties, particularly using varieties resistant
or tolerant to nematode damage during the initial phases of land use for
cropping, is an important part of taking advantage of the potential of these
resident natural enemies. Agriculturists have large amounts of capital
invested in land, equipment, and cropping costs, and consequently require a
certain degree of reliability in pest control measures. Because of the
variable nature of natural suppressiveness of nematodes, any natural control
of nematodes in the foreseeable future is most likely to arise fortuitously
rather than result from any deliberate actions by scientists or farmers
(Stirling 1991). Where soils are not naturally suppressive to nematode
populations, they may be manipulated to enhance what natural control agents
are present. Most attention in this arena has been given to the addition of
organic matter to the soils. Much of the information regarding the effects of
these amendments is circumstantial, but the beneficial effects appear
widespread. Many different soil amendments have been considered and
evaluated, and the reduction of plant damage from nematodes following such
amendments may occur through a variety of mechanisms (Stirling 1991).

One way is through the general
improvement of soil structure and fertility. Addition of crop residue or
animal manures increases ion exchange capacity of the soil, chelates
micronutrients to make them accessible by the plant, and adds available
nitrogen. Grown under such improved conditions, plants are better able to
tolerate damage from nematodes. Certain amendments may directly improve plant
resistance to nematodes (Sitaramaiah and Singh 1974). Others may contain or
release compounds which adversely affect nematodes. Among amendments
containing such compounds are those of neem (Azidiracbta indica A.
jussien) seeds or leaves and of castorbean (Ricinus communis L.)
(Stirling 1991 and references therein). Other amendments release nematicidal
compounds during decomposition. The most widely studied of these compounds is
ammonia. Because nitrogen is a constituent of nearly all soil amendments,
ammonia is usually produced during decomposition. A careful lialance must be
maintained in the carbon:nitrogen ratio, together with sufficient
concentrations of ammonia, to provide optimal effect without phytotoxicity
(Stirling 1991).

Finally, there is the direct
stimulation of nematophagous or antagonistic organisms. Spores of many
nematophagous fungi fail to germinate in otherwise suitable but nonamended
soils (Dobbs and Hinson 1953), and this soil mycostasis can affect both
spores and mycelia (Duddington et al. 1956ab, 1961; Cooke and
Satchuthananthavale 1968). Before predation of nematodes can take place,
mycelial growth and trap formation must occur. The addition of organic matter
provides a substrate that may stimulate spore germination. Organic amendments
stimulate a broad range of soil microorganisms, so the effects of amendments
on populations of these organisms are complex. Microbial population growth
generally increases immediately following the addition of organic matter and,
subsequently, as pan of the community succession, there is an increase in
populations of nematode-trapping fungi. The general hypotheses regarding the
beneficial effects of organic amendments center around the stimulation of the
saprotrophic growth phase of nematophagous fungi, and stimulation of other
general microorganisms which may be detrimental to nematodes, such as
antibiotic producing bacteria. A general rise in enzymatic levels also occurs
following soil amendment, and the enzymes may attack the structural proteins
in nematode cuticle or egg shell. Chitin amendments in particular have
received attention, and addition of chitin to soil is followed by a
relatively long-term (4-10 weeks) rise in chitinase activity in the soil.
Chitin is the principal structural component of nematode eggshells, and the
increase in chitinase activity may be accompanied by decreased survival of
nematode eggs. However, the decomposition of chitin also releases ammonia,
which may contribute to its beneficial effects. Speigel et al. (1988, 1989)
concluded that the beneficial effects of chitin amendments resulted from the
action of specialized microorganisms.

A current limitation of the
implementation of amendments for nematode control is that such amendments
must be applied in large amounts, between 1-10 tons/ha to be effective. The
use of local resources for such amendments will keep transport costs minimal.
One product, the chitin-based Clandosant (derived from crab shells), has been
marketed commercially. There is some evidence that the effectiveness of
certain amendments may be enhanced by inoculating them with degradative
microorganisms (Galper et al. 1991), and Stirling (1991) suggests
consideration of systems in which amendments can be inoctitated with a
specific microorganism as they are applied to the soil.

Augmentation of antagonists of
plant disease organisms can generally be of two types, inoculation and
inundation. Inoculative releases consist of small amounts of inoculum, with
the intention that the organisms in this inoculum will establish populations
of the antagonist which will then increase and limit the pathogen population,
In inundative releases, where large amount of inoculum is applied, with the
expectation that control will result directly from this large initial
population with limited reliance on subsequent population growth. Biological
control of plant pathogens may also rely on a hybrid of these two concepts. A
large amount of inoculum must be applied, both to increase the population of
the antagonist and to improve its distribution to favor biological control.
Also, antagonism can result from both these applied organisms and the
increased population of antagonists resulting from their reproduction.
Biological control of blackcrut (Phyllachora huberi) on rubber tree
foliage by the hyperparasites Cylindrosporium concentricum and Diicyma
pulvinata (Junqueira and Gasparotto 1991) is one example of long-term
control of a plant pathogen by a single augmentation in an agricultural
system (Cook 1993) In this case, rubber trees were treated with spore
suspensions of the antagonists (inundatively), which resulted in control over
more than one season. More generally, beneficial microorganisms are added
seasonally or more frequently.

Where the beneficial
organisms involved are being placed into a habitat or environment other than
where they originated, the organisms are often referred to as
"introduced" in plant pathology (Andrews 1992; Cook 1993). There
are several examples where such organisms, when moved to a new habitat (for
instance, from the soil to the above-ground part of a plant) colonize and
serve as successful agents of biological control (Andrews 1992; Cook 1993).

Root Diseases. One
way in which flora may be manipulated to protect against disease is to intentionally
inoculate soils or seeds with microbial antagonists. Such antagonists, to be
successful in their task, must be able to colonize plant surfaces and survive
in the competitive environment of the soil. Flora with demonstrated ability
to achieve this under field conditions include fungi, principally Trichoderma
spp., and, among the bacteria, Bacillus spp. and Pseudomonas spp.

Among the bacteria, species of Bacillus
are regularly used for biological control of root diseases, Members of the
genus have advantages, particularly that they form spores which permit simple
storage and long shelf life, and they are relatively easy to inoculate into
the soil. However, the consequence of this biology is that although the
inoculants may be present in the soil, it may be in dormant or resting
stages. Nonetheless, species of Bacillus have provided good control on
some occasions. Capper and Campbell (1986) showed a doubling of wheat yield
over wheat plants naturally infected with take-all by those also inoculated
with Bacillus pumil Meyer and Gottheil. Bacillus pumilus and B.
Subtilis were also used to protect wheat from diseases caused by species
of Rhizoctonia (Merriman et al. 1974). A major difficulty with the use
of Bacillus spp. is that the control provided is often variable, with
different results in different locations. or even in different parts of a
season in the same location (Campbell 1989). Bacillus subtilus is used
as a seed inoculant on cotton and peanut (Arachis hypogaea L.) with
nearly 2 million ha. treated in 1994 (Blackman et al. 1994). Treatment
promotes increased root mass, modulation, and early emergence, and suppresses
diseases caused by species of Rhizoctonia and Fusarium.

Of much more promise as
antagonists of root diseases are species of Pseudomonas, particularly the Pseudomanas
fluoresce and Pseudomonas putida (Trevisan) Migula groups
(Campbell 1989). These bacteria are easy to grow in the laboratory, are
normal inhabitants of the soil, and colonize and grow well when inoculated
artificially. They produce a number of antibiotics as well as siderophores.
Several have received patents and are marketed commercially for control of
root rot in cotton (Campbell 1989). An isolate of another species of Pseudomonas
has been used as anantagonist of take-all disease of wheat (Welterl983).
Isolates of Ps.fluorescens from soils showing some control of take-all
can be applied as seed coats and inoculated into fields suffering from the
disease. Such treatments give 10-27% yield increases compared with untreated,
infected control groups. Evidence points to both siderophore and antibiotic
production as important.

Species of the fungal genus Trichoderma
can be saprotrophic and mycoparasitic and have been used against wilt
diseases of tomato, melon, cotton, wheat, and chrysanthemums. The antagonists
were applied to seeds or through a bran mixture incorporated into the
planting mix at transplanting. Although disease did develop, it did so much
more slowly than in untreated soils, resulting in a 60-83% reduction in
disease (Siven and Chet 1986). The mode of action against Verticillium
albo-atrum Reinke and Berthier wilt of tomatoes appeared to be
antibiosis.

Stem Diseases. The control of Heterobasidion annosum,
the causative agent of butt rot in conifer stumps, by Pbanerochaete
gigantea was one of the first commercially available agents for
biological control of a plant pathogen (Campbell 1989). The disease caused by
H. annosum is primarily a disease of managed plantations. The fungus
colonizes freshly cut stumps, invades the dying root system and can then
infect nearby trees through natural root grafts, causing death of the trees.
However, Heterobasidion annosium, is a poor competitor, and when a
stump is intentionally inoculated with Ph. gigantea (and usually with
chemical nitrogen sources which encourage growth of the antagonist) the
antagonist rapidly colonizes the resource, excluding future attack by the
pathogen and even eliminating existing pathogen infection (Table 12.1). Very
little inoculum is needed on a freshly-cut stump, and the shelf life of the
pellet formulation is about two months at 22'C. The antagonist is able to
outcompete H. annosum even when the initial inoculum favors the
pathogen by as much as 15:1 (Rishbeth 1963).

The ascomycete fungi Eutypa
armeniaceae Hansford and Carter and Nectria galligena Bresadola
& Strass infect apricots and apples, respectively, and cause stem cankers
and eventual death of the trees. Pruning wounds in apricots are treated with Fusarium
laterium Nees:Fries through specially adapted pruning cutters. Fusarium
laterium produces an antibiotic which inhibits germination and growth of E.
armeniaceae. When applied, the concentration of the antagonist must be
greater than106 conidia/ml. Integrated application which includes a
benzimidazole fungicide gives better control than either fungicide or
antagonist alone. Nectria galligena infection can be reduced through
sprays of suspensions of Bacillus subtilis or of Cladosporium
cladosporioides (Fresenius) de Vries. These antagonists are not in
commercial use because apples are treated for Venturia inaequalis (Cooke)
G. Winter (apple scab) so frequently that V. galligena is controlled
by those sprays.

Crown gall is a stem dlisease caused
by the bacterim Agrobacterium tumefaciens (Smith & Townsend) Conn.
It affects both woody and herbaceous plants in 93 families. Infection is
typically from the soil, rhizosphere, or pruning tools. Control can be
effected by treating plants with a suspension of a related saprotrophic
bacterium Agrobacterium radiobacter (Beijerinkand van Delden)
Conn strain K-84. This strain of the bacterium produces an antibiotic that is
taken up by a specific transport system in the pathogen bacterium, which is
then killed. The commercially available formulations of this agent are
effective primarily against pathogen strains which attack stone fruits, but
other bacteria are under investigation for use against strains pathogenic in
other crops. This agent has been altered by gene-modifying technology to
produce a new strain (strain 1024) which lacks the ability to transfer
antibiotic resistance to the target bacterium (Van Driesche & Bellows
1996).

The fungus Cbondrostereum
purpureum (Persoon: Fries) Pouzar infects stems of fruit trees and
produces a toxin which leads to a condition known as silverleaf disease.
Stems can be inoculated with a species of Trichoderma grown on wooden dowels
or prepared as pellets which are inserted into holes bored in the affected
stem. Treated stems recover from the disease more rapidly than untreated
stems. The Trichoderma sp. can be applied to pruning wounds to prevent
initial establishment of C. purpureum.

Leaf Diseases. Control of leaf diseases at the time of
pathogen germination has been demonstrated in the laboratory. This control
occurs in the presence of competitive organisms, which may include fungi,
yeast, or bacteria. The mode of action in some cases is competition for
nutrients that, together with water, are necessary for successful germination
and invasion of many pathogens. The germination of Botrytis sp., for
example, is inhibited by certain bacteria and yeasts (Blakeman and Brodie
1977). This inhibition is less pronounced if additional nutrients are
supplied, indicating that the mechanism is, at least in parrt, resource
competition. Studies on control of Botrytis rot in lettuce (Wood 1951)
indicated that several organisms were successful in suppressing the disease
when sprayed on lettuce plants, among them species of Pseudomonas,
Streptomyces, Ttichoderma viride, and Fusarium. Peng and Sutton (1991)
evaluated 230 isolates of mycelial fungi, yeasts, and bacteria and tested
them as anntagonists of B. cinerea in strawberry in both laboratory
and field trials. Several organisms (including members of each taxonomic
group tested) were effective, some as effective as captan (a commercial
fungicide). Sutton and Peng (1993b) further evaluated Gliocladium roseum and
determined that the suppression of B. cinera by this antagonist was
probably a result of competition for leaf substrate. The fungi Gliocladium
roseum and Myrothecium verrucaria (Albertini and Schweinitz)
Ditmar were also effective in suppressing B. cinerea in black spruce (Picea
mariana [Miller] Britton Stearns Poggenburg) seedlings (Zhang et al.
1994).

Bacteria may also be used to limit
frost damage to leaves and blossoms of plants. Certain bacterial species such
as Pseudomonas syringae and Erwinia herbicola serve as
nucleation sites on leaves for the formation of ice, and, in their presence,
ice forms soon after temperatures fall below freezing. If these ice
nucleating bacteria are replaced by competitive antagonists (such as certain
strains of Ps. syringae) that lack the protein that causes ice
nucleation, frost is prevented even at temperatures from -2 to 5E C.(1 (Lindow 1985b). The protective
bacteria, after being applied it) the leaves, colonize them for up to two
months, an interval suitable to protect from frost during the limited season
that low temperatures are likely. A naturally-occurring, non-ice nucleating
strain of Ps. fluorescens is registered in the United States as a
commercial product (Frostban B ) for suppression of frost damage (Wilson and
Lindow 1994).

Spraying suspensions of
propagules, generally at high concentrations, is the principal method for
applying biological control agents to foliage (and to flowers), and dusts
(such as lyophilized bacterial preparations) are also used. Spray methodology
has yet to be refined in terms of sprayer characteristics, droplet size, and
pressures, and other methods of application with greater efficiency may be
necessary to effectively target certain plant parts (Sutton & Peng
1993a).

Flower Diseases. A principal disease of flowers, which has
received attention, is fire blight of rosaceous plants, which is particularly
severe on pear (Campbell 1989). The causal bacterium, Erwinia amylovora, also
occurs on leaves and may cause stem cankers. Insects transfer the bacterium
to flowers in the spring from overwintering sites on stem cankers, and
subsequently from flower to flower. Infection enters the pedicel and from
there the stem. Infected flowers and small stems die, and cankers form on
other stems. Chemical control is difficult and expensive, and sometimes is
ineffective because of resistance to copper compounds and streptomycin.
Biological control has been effective using E inia berbicola, sometimes
in combination with Pseudomonas syringae (Wilson and Lindow 1993).
Suspensions of E. berbicola are sprayed onto the flowers just before
the period of potential infection. The antagonist occupies the same niche as
the pathogen, reducing the numbers of E. amylovora by competition, and
there is also evidence for the production of bacteriocins (chemicals which
suppress population growth of related bacteria) by some strains. Control can
be good, comparable to that achieved by commercial bactericides, though
repeated application of the bacterium was necessary (Isenbeck and Schultz
1986). Another approach to control is to reduce secondary infections on
leaves, which leads to reductions in the overwintering population of the
pathogen. This control is achieved by treatment with the antagonists
Ps.syringae and other bacteria (Lindow 1985b). A novel approach to
dissemination of the antagonistic bacteria has been evatuated by Thomson et
al. (1992). They mixed E. herbicolaancl Ps. fluorescens with pollen in
a special apparatus at the entrance to honey bee (Apis mellifera) hives.
Bees emerging from these hives through the mixtures transmittecl the
antagonists to the flowers efficiently, although disease control was not
evaluated because of absence of disease in the test orchards.

Fruit Diseases. Fruits are subject to attack both by
general pathogens (Botrytis, Rhizopus, Penicillium) and by a few
specialist pathogens such as the coffee berry disease fungus Colletottichum
coffeanum Noack and Monilinia spp., which cause brown rots of
rosaceous fruits. While many of these are controlled by fungicides, Trichoderma
viride has been shown to limit disease from Monilinia spp. Various
Bacillus spp. also are antagonistic to these fungi through production of
antibiotics and by reducing the longevity and germination of spores. Both the
bacteria and culture filtrates have been used with some success against these
pathogenic fungi, but there has been no commercial development, probably
because fungicides used routinely in orchards for control of other diseases
give some control of brown rot (Campbell 1989). Among the most serious
diseases of soft fruits are postharvest rots (Dennis 1983), especially that
caused by Botrytis cinerea. Potential for biological control of
postharvest diseases was reviewed by Wilson and Wisniewski (1989) and
Jeffries & Jeger (1990) (also see Wilson and Wisniewski 1994). In
strawberries, B. cinerea grows saprotrophically on crop debris and
from there infects flowers or fruit. Various species of Trichoderma have
been evaluated and gave control as good as standard fungicides (Tronsmo and
Dennis 1977). The antagonists Clado,@Cladosporium herbarium and
Penicillium sp. gave excellent results in controlling Botrytis rot
on tomato (Newhook 1957). Honey bees have been used to distribute Gliocladium
roseum to strawberry flowers (Peng et al. 1992) and raspberry flowers
(Sutton and Peng 1993a) to suppress Bot?ytis rot.

Root Diseases. Induced resistance is a form of biological
control in which the natural defense responses of the plant, which may
include production of phytoalexins, additional lignification of cells, and
other mechanisms (Horsfall and Cowling 1980; Bailey 1985), are promoted in
the plant prior to exposure to the pathogen (Van Driesche & Bellows
1996). Challenging the plant with a nonpathogenic organism induces these
resistance mechanisms. The induced plant defenses then limit later infection
by the pathogen. The organism employed may be an avirulent strain of the
pathogen, or a different specialized form, or even a different species, There
are few well-documented cases of induced resistance for soil-borne pathogens,
and these are mostly of wilt diseases. Dipping tomato roots in a suspension
of Fusarium oxysporumf.sp. dianthi a few days before likely
exposure to the pathogen F oxysporum f.sp. lycopersici (Saccardo)
Snyder and Hansen conferred protection that lasted a few weeks. Cotton may be
protected for three months or longer by spraying the roots at transplanting
with a mildly pathogenic strain of the disease causing pathogen Vcrticillium
albo-atryum. The role of some fungi against take-all of wheat includes
some elements of induced resistance. Gaeumannomyces graminis var. graminis
grows on grass roots and also has been found on wheat, where it occupies a
niche similar to that of the pathogen G. graminis var. tritici Walker.
The antagonist invades the root cortex but not the stele, and is halted by
the lignification and suberization of the cortex and stele. Root cells with
these chemically-changed walls are less susceptilile to invasion by the
pathogen. Although this interaction produced yield increases in Europe, the
strains or species present in the United States did not appear to confer
resistance, and in Australia there were only slight yield increases (Campbell
1989). These variable results, while somewhat common for biological control
of soil borne pathogens, do not reduce the value of the antagonists where
they do work, but rather indicates some potential challenges in defining the
taxonomy, biology, and host-plant relationships important to biological
control in this group of organisms.

Leaf and Stem
Diseases. Induced
resistance can control anthracnose diseases caused ny Colletotrichum spp.
(Kúc 1981; Dean and Kúc 1986). Colletotricbum lindemthianum (Saccardo
& Magnus) Lamson Scribner causes anthracnose of beans, Colletotrichum
lagenarium (Passerine) Saccardo causes cucumber anthracnose, and Cladosporium
cucumerinum Ellisand Arthur causes scab in cucumbers. Inoculation
of cucumbers with Colletotrichum lindemuthianum (which does not cause
disease in cucumbers) made plants resistant to both Colletotrichum
lagenarium and Cladosporium cucumerinum. Treatment applied to an
early leaf resulted in protection of later leaves, even when the initially
inoculated leaf was removed. The factor causing resistance travels
systemically through the plant. Variations on this approach include
inoculating an early leaf with a pathogen, inducing resistance throughout the
plant, and then removing the infected leaf. Induced resistance also occurs in
some virus diseases (Thomson 1958) and may last for years, as in the case of
healthy citrus seedlings being inoculated with an avirulent strain of citrus
tristeza vinus

Stem rot in carnations, caused by Fusarium
roseum Link: inoculating wounds inflicted during propagation with the
nonpathogenic F. roseum 'Gibbosum' can prevent Fries 'Avenaceum,'.This inoculation produced a germination inhibitor and also reduced the
time needed for the stems to develop resistance to the pathogen. This
hastening of resistance was caused by activation of the host's defense
mechanisms, and is another example of induced resistance.

Employing hypoviruient strains of
the disease pathogen controls chestnut blight, caused by Cryphonectria
parasitica. A number of hypovirulent strains are known, and inoculating
infected trees with a hypovirulent strain leads to reduced canker size and
greater stem survival. In the field, hypovirulent strains are inoculated into
infected trees at the rate of 10 inoculated trees/ha. The hypovirulent strain
spreads from these locations and, on contacting more virulent strains, fuses
with these strains and exchanges a viral element infecting the pathogen (Van
Driesche & Bellows 1996). The hypovims, which causes hypovirutence, is
transferred to the virulent strains, attenuating their effects. Active
cankers are eliminated in 10 years (van Alfen 1982).

Root Diseases. The mycoparasites Tiichoderma spp,
have been used successfully against diseases caused by Rbizoctonia and
Sclerotium pathogens. One example is the pathogen Sclerotium
rolfsiii Saccardo, which attacks many crop plants and survives
unfavorable periods by forming sclerotia in the soil. Strains of T barzanium
that have Beta 1-3
glucanases, chitinases, and proteases have been isolated. These enzymes
permit T barzanium to parasitize the hyphae and sclerotia of the
pathogen, invading and causing lysis of the cells. Trichoderma harzanium is
grown on autoclaved bran or seed, and this material is then mixed with the
surface soil (Chet and Henis 1985). Two other fungi known to parasitize
sclerotia are Coniothryium minitans and Sporidesmium sclerotivorum (Ayers
and Adams 1981).

Sporidesmium sclerotivorum is
a hyphomycete that in nature behaves as an obligate parasite of sclerotia of Botrytis
cinerea and several species of Sclerotium (Adams 1990). It has
been studied as an agent against botrytis rot in lettuce, where it shows
considerable potential. It can be grown in vitro on various carbon
sources and is efficient in converting glucose into mycelium. Spores produced
in mass culture are collected, processed, and applied to infected soil, and
field tests are promising (Adams 1990).

Leaf Diseases. Some plant pathogens, including fungi and
some bacteria, are known to be attacked by other pathogens. Bdellovibrio
bacterivorus is a bacterium that can attack other bacteria by penetrating
the cell wall and lysing the host bacterium, subsequently reproducing inside
its host. Different strains of Bd. bacterivorus have been examined for
virulence against Pseudomonas syringae pv. glycinae (Coeper) Young,
Dye and Wilkie, the cause of soybean blight. By applying Bd. bacteriovorus
at sufficiently high rates, disease symptoms were reduced more then 95%
(Scherff 1973). Parasites of fungi pathogenic on leaves are numerous (Kranz
1981), but only a few have been studied in much detail, such as Sphaerellopis
filum, Verticillium lecanii and Ampelomyces quisqualis. The
mycoparasite typically penetrates the host hypha or spore and kills it. Some
of the control may be from the pathogen overgrowing the sporulating pustules
of the pathogen and preventing spore release and thus reducing inoculum in
the environment, even if the spores are not killed. A typical problem with
implementation of these mycoparasitic fungi is that they often do not affect
a large proportion of the pathogens unless humidity and temperature are high.
Consequently, although much reduction of spore production may take place,
there is still sufficient inoculum of the pathogen remaining to cause
disease. These mycoparasites often are seen only at high incidences of
disease, which is unsuitable for general control of the target pathogens.
They may have some use in particular systems, either in the tropics or in
greenhouses, where environmental conditions are more favorable.

Plant-Parasitic
Nematodes. The
bacterial pathogen of nematodes most studied is Pasteuria penetrans sensti
stricto Starr and Sayre (Starr and Sayre 1988), which is an obligate
parasite of root-knot nematodes (Meloidogyne spp.) and has not been
successfully cultured in vitro. This restriction in mass culturing has
limited attempts to test the bacterium's effectiveness (Stirling 1991). In
experimental trials, it has shown potential for controlling root-knot
nematodes (Meloidogyne spp.) (Mankau 1972; Stirling et at. 1990),
infesting a high proportion of nematodes in soil to which bacterial spores had
been added, and in other trials (U. S. Department of Agriculture 1978)
reducing damage to plants in plots containing the bacterium. Observations by
Mankau (1975) indicated that populations of the bacterium did not increase
rapidly in field soil. The development of a mass production method in which
roots containing large numbers of infected Meloidogyne spp. females
were air-dried and finely ground to produce an easily handled powder enabled
more extensive testing (Stirling and Watchel 1980). When dried root
preparations laden with bacterial spores were incorporated into field soil at
rates of 212-600 mg/kg of soil, the number of juvenile Meloidogyne.
javanica (Treub) Chitwood in the soil and the degree of galling was
substantially reduced (Stirling 1984); other authors have reported similar
results (Stirling 1991). Effective use of this bacterium through such
inundative release would require concentrations on the order of 105 spores/g
soil (Stirling et al. 1990). Such quantities could only be produced on a
large scale with an efficient in vitro culturing method, a problem
which has received attention but has not yet yielded a solution (Stirling
1991). Use in inoculative releases, where smaller numbers of spores are
applied and a crop tolerant of nematode damage is grown to permit the
increase of both nematode and bacterial populations, has been suggested
(Stirling 1991). Conserving the bacterium in the presence of nematicides
appears possible. Of seven tested nematicides, only one showed slight
toxicity to the bacterium (U.S. Department of Agriculture 1978). The use of Bacillus
thuringiensis strains with activity against nematodes is also possible.
As these bacteria may be cultured in fermentation media. their mass culture
is simpler than forP penetrans. Suppression of nematodes was possible
through drench applications and through incorporating the bacterium into a
methyl cellulose seed coat (Zuckerman et al. 1993).

Considerable attention has been
given to the nematode-trapping fungi as possible augmentative agents, Mass
culture on nutrient media is possible for these fungi. Two cultures of
nematophagous Arthrobotrys fungi have been developed and tested for
addition to soil for specific target environments. Cayrol et al. (1978)
reported the successful use of Arthrobotrys robusta Cooke and Ellis
var. antipolis, commercially formulated as Royal 300 against the
mycetophagous nematode Ditylenchus myceliophagus Goodey in commercial
production of the mushroom Agagaricus bisporus (Lange) Singer. The
nematophagous fungus was seeded simultaneously with A. bisporus
mushroom compost, which led to 280/o increases in harvest and reduced
nematode populations by 40%. The results justified the commercial use of the
fungus for nematode control in mushroom culture, Cayrol and Frankowski (1979)
reported the use of Arthrobotrys superba Corda (Royal 3509) in tomato
fields, applied to the soil at a rate of 140 g/M2, resulting in protection of
the tomatoes and colonization of the soil by the fungus. Other reports have
indicated little efficacy of fungal preparations when added alone to soil
(Barron 1977; Sayre 1980; Rhoades 1985). In general, there has been limited
success in the use of these agents (see Stirling [1991] for a summary). The
fungistatic nature of soil (Mankau 1962; Cooke and Satchuthananthavale1968)
may limit the ability of these fungi to grow even when added in substantial
numbers to soil. Additional work is needed, perhaps in the areas of
colonization and soil amendments together for the use of nematophagous fungi
to become suitably reliable for general use as a control method (van Driesche
& Bellows 1996).

Many predacious fungi may be
unsuited for control of root-knot nematodes, Meloidogyne spp. Stirling
(1991) suggested that Monacrosporium lysipagum (Drechsler) Subramanian
and Monacrosporium ellipsosporum (Grove) Cooke and Dickinson, which
can invade egg masses, may warrant further investigation. The
nematode-trapping fungi are likely to be more effective against ectoparasitic
nematodes and such species as Tylenchus semipenetrans Cobb, where
juvenile stages migrate through the rhizosphere. Little attention has been
given to testing predacious fungi against such nematodes (van Driesche &
Bellows 1996).

Fungi which are internal parasites
of nematodes are difficult culture on nutrient media, and consequently there
have been few attempts to use them for augmentative control of nematodes.
Alternative mass-culturing techniques may hold promise (Stirling 1991). In
the few experiments reported, the fungistatic effects of soil often limited
fungal growth and the effectiveness of the antagonists. Lackey et al. (1993)
report the production and formulation of Hirsutella rhossiliensis Minter
et Brady on alginate pellets (see also Fravel et al. 1985) which, when added
to soil, led to transmission of the fungus to the nematode Heterodera
schachtii Schmidt and suppressed nematode invasion of roots.

Among the facultatively parasitic
fungi which attack nematodes, Paecilomyces lilacinus and Verticillium
cblamydosporium have received the most attention as possible augmentative
agents. The results of studies on P lilacinus have been variable, with
some studies showing some positive effect of the fungus, while others show
little or no effect (Stirling 1991). The mechanisms leading to the beneficial
effect have not been clearly elucidated, but may be from metabolic products
or effects other than direct parasitism of eggs. Studies have generally
involved the addition of fungal preparations to the soil at the rate of 1-20
tons/ha, which is really too great for widespread commercial use. Additions
at lower rates (0.4 tons/ha) in a variety of carriers (alginate pellets,
diatomaceous earth, wheat granules) have also shown limited beneficial
effects (Cabanillas et al. 1989; Stirling 1991). Tribe (1980) suggested the
direct addition of V. Chlamydosproium to the soil. Kerry (1988) added
hyphae and conidia, formulated in sodium alginate pellets or in wheat bran,
to soil, and the fungus proliferated in the soil only from granules
containing bran. When chlamydospores were used as inoculum, the fungus was
able to establish without a food base (De Leij and Kerry 1991). Of three
isolates studied, only one successfully colonized tomato root surfaces. This
species apparently has considerable promise, but screening programs will be
necessary to identify isolates with characteristics suitable for biological
control (Stirling 1991).

Predacious microanhropods and
nematodes have evoked considerable interest. Most work has been done in
simple microcosms, and there have been no attempts to evaluate augmentative
release of these organisms in a field setting. In one experiment, Sharma
(1971) found nematode numbers reduced by 50% or more in glass jars inoculated
with mites and springtails compared with similar jars containing no
predators, but the author pointed out possible causes for the reduction other
than simple predation. Experiments with predacious nematodes have in general
failed to demonstrate a measurable impact of the predator (Stirling 1991).
One exception was the reduction of galling by Meloidogyne incognita on
tomato by predacious nematodes (Small 1979). The general suitability of these
groups of organisms for inundative release is questionable, because of the
potential difficulties in developing technologies for their rearing,
packaging, transport, and delivery beneath the soil in a viable state
(Stirling 1991).

Mycorrhizae are nonpathogenic
fungi associated with roots in some temperate forest trees. Ectomycorrhizae
are mostly basidiomycetes which form a sheath over the root, and hyphae
spread out into the soil. These fungi have been studied in relation to
nutrient uptake, but they also affect root disease. Because they completely
enclose the root, they change the quantity and quality of exudates reaching
the soil; consequently, roots with mycorrhizae have a different rhizosphere
flora than uninfectect roots (Campbell 1985). In at least one case, the
mycorrhizal fungus Pisolitbus tinctorius (Persoon) Coker and Couch,
the thick symbiont sheath forms a barrier to infection by such pathogens as Phytophthora
cinnamomi attacking eucatyptus trees. Other mycorrhizal fungi produce
antibiotics effective against P cinnamomi in plate tests. The
intentional manipulation of mycorrhizal fungi for disease control has not
been widely implemented, but opportunities for selected uses may be possible
(Campbell 1989).

Another group of fungi are the
vesicular arbuscular mycorrhizae (VAM), which are phycomycete fungi
associated with the roots of many plant species including many crops. These
fungi do not form a sheath surrounding the root, and their effects on disease
are complicated, but are in general beneficial (Campbell 1989). Some of these
effects may involve changes in host plant physiology in the presence of the
symbiont, as there is no direct evidence of pathogen inhibition by these
fungi.

Growth of our knowledge about biological
control of plant diseases has been extensive since the first experimental
reports (Hartley 1921; Sanford 1926; Millard,,ind Taylor 1927; Henry 1931),
and substantial potential for microbial control of pathogens has been
demonstrated. A number of products or programs have reached the stage of
commercial development or availability (van Driesche & Bellows 1996).
Products in current use include both those aimed at specialty markets for
control of certain stem or flower diseases (for which chemical control is
either unavailable or expensive) and those aimed at larger scale markets such
as seed treatments for widely planted crops.

The cycle for research,
development, and implementation of antagonists of plant pathogens is composed
of several steps. These include initial discovery of candidate agents,
refinement of knowledge of their biology, ecology, and mode of action,
microcosm and field trials of their efficacy, and large-scale development for
commercial production.

The first challenge in the
development of a biological control program is the discovery process. Many
microorganisms show potential as antagonists of particular pathogens.
Protocols have been proposed to make the process of screening these
candidates more efficient (Andrews 1992; Cook 1993). The principal
difficulties are screening out candidates that are effective only during in
vitro (agar plate) trials but are not effective in natural settings, and
in selecting candidates that can be successfully cultured in large quantities.
Following discovery of suitable candidates, research focuses on their mode of
action and on factors which may enhance or limit their efficacy in targeted
settings (glasshouses, field plots). In addition, experimental fermentation
and formulations must be developed for production of materials suitable for
use in agricultural settings. Finally, issues of large-scale production and
delivery must be addressed. Products for use must be effective on an
economical basis, and economies of scale may play an important role in the
eventual availability of any organism or product. Each must have a
satisfactory shelf life, and safe and effective methods for application must
be discovered or developed (Cook 1993; Sutton and Peng 1993a). Such
application methods might include sprays of suspensions or dusts, contact
application, honeybee and other bee vectoring, and production of antagonists
in a crop environment (Sutton and Peng 1993a).

Adopting any biological control
agent in commercial agriculture is dependent on its reliability and
availability. Limitations to the process of eventual adoption, therefore,
include cost of development and size of potential market (van Driesche &
Bellows 1996). Many pesticides for control of plant diseases have a broad
spectrum of activity, are applicable in a variety of crops and settings, and
may act either prophylactically, therapeutically, or both. Biological
controls, in contrast, often have narrow ranges of activity and may work in
only a few crops or soil types, and while they can often act both as a
barrier and therapeutically, their action may take some time to develop.
Therefore, they may have a narrower market than a chemical pesticide and be
unattractive for development by major corporations (Andrews 1992). In this
context, it may be appropriate for public institutions such as government
experiment stations to undertake the development of such biological controls,
in the same way that they take the responsibility for development of new
plant varieties (Cook 1993).

Those microorganisms intended for use as biological control agents
must be viewed in a biological rather than a chemical control (Cook 1993).
Where an effective pesticide may work in many places, each place may have
unique edaphic, and biological features which limit or enhance the
effectiveness of microbial antagonists of pathogens. Consequently, each
microbial biological control system may have to make use of locally adapted
strains, taking advantage of resident antagonistic flora and fauna and augmenting
their effectiveness with additional species or strains, or enhancing resident
populations through soil amendments. Although the different strains may use
common mechanisms to achieve biological control (such as production of
antibiotics), competitive abilities adapted to local conditions may be vital
to permit the organisms to compete for resources and effectively control
pathogens (van Driesche & Bellows 1996)